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Biomedical subjects

G Colombo

Publications and source records attributed to G Colombo.

At least 199 records · Page 11Linked to original sources
PubMed1984

Kinetic and spectroscopic studies of the interaction of copper with dopamine beta-hydroxylase.

The question of the stoichiometry of copper bound to dopamine beta-hydroxylase and the number of copper atoms required for maximal activity was addressed in this study. Incubation of tetrameric enzyme from bovine adrenal medulla with 64Cu2+ followed by rapid gel filtration yielded an enzyme containing 8.3-8.9 mol of Cu/mol of tetramer. An identical stoichiometry was obtained by analysis of bound copper by atomic absorption methods. NMR and EPR were used to monitor titrations of the enzyme with Cu2+ and showed that the longitudinal relaxation rate of solvent water protons and the amplitude of the signal at g approximately 2 increased linearly up to a copper to protein ratio of approximately 8. Additional titrations also indicate that an enzyme-Cu2+-tyramine-CN- inhibitory complex was formed when 8 mol of Cu2+ are bound per mol of enzyme. The rate of inactivation of dopamine beta-hydroxylase by the mechanism-based inhibitor 2-Br-3-(p-hydroxyphenyl)-1-propene was measured and used as a method to follow enzymatic catalysis. An increase in rate was observed with increasing Cu2+ up to a protein to Cu2+ ratio of 8 Cu/tetramer. The rate becomes constant after this ratio is achieved. These data indicate that dopamine beta-hydroxylase specifically binds 8 mol of Cu/tetramer and that this stoichiometry is required for maximal activity.

Adrenal Medulla↗
PubMed1984

Lysosomal glycohydrolases in normal T and non-T peripheral lymphocytes.

The optimal assay conditions and the levels of seven lysosomal glycohydrolases (alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucosidase, beta-D-glucuronidase, beta-N-acetyl-D-glucosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase), alpha-D-mannosidase, alpha-L-fucosidase) were determined in human peripheral unseparated lymphocytes, T and non-T lymphocyte subpopulations. From fifteen adult volunteers the enzymes were assayed by fluorimetric procedures using the corresponding 4-methylumbelliferyl glycosides as substrates. The enzyme assay procedures displayed good precision and reproducibility. All the tested enzymes had higher activities in non-T than T lymphocytes. This difference was statistically highly significant, especially when the enzyme contents were expressed on a DNA, rather than mg protein, basis. Unseparated lymphocytes displayed levels of lysosomal enzymes which corresponded to the proportion of T and non-T lymphocytes in the unseparated preparation, indicating that the process of lymphocyte fractionation caused neither loss nor activation of lysosomal enzymes. It is concluded that the observed difference in lysosomal enzyme levels is an authentic imprint of the two lymphocyte subpopulations, implying a differential role played by the lysosomal apparatus in the same cells.

Adult↗
PubMed1984

Alternate substrates of dopamine beta-hydroxylase. III. Stoichiometry of the inactivation reaction with benzyl cyanides and spectroscopic investigations.

Dopamine beta-hydroxylase was incubated with p-hydroxybenzyl cyanide, ascorbate, and O2 and the products of the hydroxylation reaction were monitored by high performance liquid chromatography. At early times, p-hydroxymandelonitrile was the sole product but this compound slowly decomposed to p-hydroxybenzaldehyde and cyanide. Dopamine beta-hydroxylase was also inhibited under these reaction conditions but the amount of cyanide produced was insufficient to account for the extent of enzyme inhibition. Incubation of dopamine beta-hydroxylase with [ring-3H]p-hydroxybenzyl cyanide, ascorbate, and O2 resulted in incorporation of detectable radiolabel into enzyme only when 100% O2 (0.4 X Km) was present. The amount of incorporated radiolabel was substoichiometric with respect to enzyme subunits (approximately 30%) and also did not correlate with the total amount of inhibited dopamine beta-hydroxylase. These data suggest two different modes of inhibition: covalent-adduct formation at early times and inhibition due to cyanide generated at the active site. Separate K14CN binding studies with dopamine beta-hydroxylase corroborated the latter suggestion since 2 mol of cyanide were bound per tetramer. Tyramine stabilized [14C]cyanide binding to dopamine beta-hydroxylase, consistent with the lack of enzyme reactivation observed under certain conditions (Colombo, G., Giedroc, D. P., Rajashekhar, B., and Villafranca, J. J. 1984) J. Biol. Chem. 259, 1601-1606). EPR data for dopamine beta-hydroxylase-bound Cu2+ directly demonstrated that a quaternary complex with tyramine and cyanide was formed, since the spectrum of dopamine beta-hydroxylase-Cu2+-tyramine-cyanide is distinct from that of dopamine beta-hydroxylase-Cu2+-cyanide and dopamine beta-hydroxylase-Cu2+-tyramine. A comprehensive mechanism for dopamine beta-hydroxylase inhibition by benzyl cyanides is presented.

Acetonitriles↗
PubMed1984

Alternate substrates of dopamine beta-hydroxylase. I. Kinetic investigations of benzyl cyanides as substrates and inhibitors.

A series of benzyl cyanide analogs have been studied as substrates and inhibitors of dopamine beta-hydroxylase to extend our initial report (Baldoni, J. M., and Villafranca, J. J. (1980) J. Biol. Chem. 255, 8987-8990) which showed that p-hydroxybenzyl cyanide was a suicide substrate of dopamine beta-hydroxylase. Thus, the appVmax values for benzyl cyanide analogs decrease in the order p-OH greater than m-OH greater than H much greater than p-OCH3,m-OCH3; the m-OH, m-OCH3 and p-OCH3 analogs are competitive inhibitors versus tyramine in initial velocity studies. The Vmax values for tyramine and p-hydroxybenzyl cyanide are nearly identical at saturating O2 and ascorbate (pH 5.0, 37 degrees C) but the Km for O2 is 0.14 and 2.8 mM, respectively, with tyramine and p-hydroxybenzyl cyanide. Studies of the pH dependence of log V/K for tyramine show two pKa values of 5.2 and 5.8 while for m-hydroxybenzyl cyanide the values are 5.3 and 5.9. The log Vmax profile shows one pKa of 5.9 with tyramine as substrate. Thus, nearly identical enzymic groups are involved in binding and/or catalysis with these two substrates. All the benzyl cyanide analogs are suicide inactivators of dopamine beta-hydroxylase. With m-hydroxybenzyl cyanide, the partition between catalysis and inactivation (kcat/kinact) changed from approximately 600 to approximately 17 as the pH varied from 5.0 to 6.7. The log kinact versus pH profile shows one pKa value of 6.0, suggesting that an enzymic group must be deprotonated for maximal inactivation. Copper was essential for the suicide inactivation of dopamine beta-hydroxylase by benzyl cyanides and kinetic studies of partially inhibited dopamine beta-hydroxylase (approximately 50%) showed that inactive enzyme molecules were completely inactive. The following papers in this series discuss the partial reactivation of suicide-inhibited dopamine beta-hydroxylase and the stoichiometry of inactivation by benzyl cyanide analogs.

Adrenal Medulla↗
PubMed1984

Alternate substrates of dopamine beta-hydroxylase. II. Inhibition by benzyl cyanides and reactivation of inhibited enzyme.

Several ring-substituted benzyl cyanides lead to inactivation of dopamine beta-hydroxylase during catalysis. With m-hydroxybenzyl cyanide, maximal inactivation occurs when an enzyme group with a pK alpha of 6.0 +/- 0.2 is ionized (Colombo, G., Rajashekhar, B., Giedroc, D. P., and Villafranca, J.J. (1984) J. Biol. Chem. 259, 1593-1600). This paper reports studies conducted to determine the stability of inactivated dopamine beta-hydroxylase. Inactivation of the enzyme by m-hydroxybenzyl cyanide at pH 6.4 is halted by lowering the pH to approximately 5.0 with acetate, fumarate, pyridine, or phosphate buffer in the presence of tyramine. However, if tyramine is omitted, reactivation occurs. The extent of reactivation is dependent upon the final pH value and buffer used to adjust the pH. Reactivation is observed as the pH is lowered from 6.4 to below 5.7 with acetate, fumarate, or HCl. With phosphate, reactivation occurs at any pH value from 6.9 to 4.5 but is greater at lower pH values. Thus, inactivation and reactivation have opposite pH dependencies. Also, reactivation is dependent upon the elapsed time of inactivation. At early times, no reactivation is observed when phosphate is used to adjust the pH, but reactivation is observed later in the inactivation reaction. Reactivation to 100% of the original activity does not occur under these conditions. These data suggest at least two inactivation mechanisms by benzyl cyanides: 1) formation of a tightly bound or covalent adduct between dopamine beta-hydroxylase and enzyme-bound mandelonitrile (or a rearranged form of this molecule), and 2) reversible inhibition resulting from cyanide binding to enzyme-Cu2+. Studies with radiolabeled p-hydroxybenzyl cyanide as well as EPR studies of dopamine beta-hydroxylase-Cu2+ are reported in the following paper.

Adrenal Medulla↗
PubMed1984

Central benzodiazepine receptors in the spinal cord and other regions of the CNS of the cat.

Central benzodiazepine (BZ) receptors were looked for in the spinal cord, cerebral cortex and cerebellum of the cat. Both [3H]Ro 15-1788 and [3H]flunitrazepam bound to benzodiazepine receptors with apparent dissociation constants (KD) in the nanomolar range and Hill coefficients near unity. The concentration of binding sites was much greater (10-40 times,depending on the ligand used) in the cortex and cerebellum than in the spinal cord. gamma-Aminobutyric acid (GABA) significantly reduced the KD value of the binding of tritiated flunitrazepam in all three areas of the central nervous system (CNS). The displacement of [3H]Ro 15-1788 by different benzodiazepine ligands indicates a relative prevalence of BZ1 (high affinity for beta-carboline esters and the triazolopyridazine, CL 218 872) in the cerebellum, a predominance of BZ2 (low affinity for the same agents) in the spinal cord and a mixture of both types in the cortex. The possibility that there is regional heterogeneity of receptors for benzodiazepines in CNS of the cat is discussed.

Animals↗
PubMed1984

Studies on the luteolytic activity of MDL-646, a new gastroprotective PGE1 analogue, in the hamster.

MDL-646, 11,15-dihydroxy-16-methyl-16-methoxy-9-oxo- prost -13-en-1-oic acid methyl ester, is one of the most active members of a new class of PGE1 analogues with potent gastric cytoprotective and antisecretory activity. The potential luteolytic activities of MDL-646 and its corresponding PGE2 derivative, L 14224 were assessed from their ability to terminate pregnancy and to reduce plasma progesterone levels in the hamster. PGE1 and PGE2 were used as reference compounds. The biological and biochemical data clearly demonstrate that these 16-methyl-16-methoxy PGE derivatives, given s.c. or p.o. either once or for 3 days, have no luteolytic effects up to a daily dose of 2-2.5 mg/kg, and are therefore at most 1/2 to 1/4 as luteolytic as the parent natural PGEs. The dissociation between gastroprotective and luteolytic activity was interpreted to indicate that these new PGE derivatives have a specific action.

Abortion, Spontaneous↗
PubMed1984

Inhibition of PG production by MDL 035, a new non-steroidal non-acidic anti-inflammatory compound, in rat gastric mucosa and inflammatory exudate.

The effects of 3-(1-methylethyl)-2-(4-methoxyphenyl)-3H-naphth [1,2-d]imidazole, MDL 035, a new non-steroidal non-acidic anti-inflammatory compound, on the production of prostaglandin (PG) in rat gastric mucosa in vivo and in vitro and in inflammatory exudate in vivo were studied. MDL 035 reduced PGE2 and 6-keto-PGF1 alpha levels more effectively in inflamed tissue than in gastric mucosa when assayed in in vivo experiments, whereas indomethacin and other non-steroidal anti-inflammatory drugs (NSAIDS) are equally effective in both systems. MDL 035 is almost as active as indomethacin when incubated with gastric tissue in vitro. The better gastric tolerance of MDL 035 than of other NSAIDS is discussed in relation to these differences in in vivo and in vitro effects.

6-Ketoprostaglandin F1 alpha↗
PubMed1983

Contragestational effects of DL-alpha-difluoro-methylornithine, an irreversible inhibitor of ornithine decarboxylase, in the hamster.

In the hamster, there are remarkable changes in ODC activity in various uterine reproductive tissues during gestation. When DL-alpha-difluoromethylornithine (DFMO), a highly selective irreversible inhibitor of ODC, is given during the early post-implantation period, when ODC activity in the conceptus is rapidly rising and reaches its highest values, it causes pregnancy arrest. Low doses of DFMO are contragestational only during a short period of gestation (day 7 and 8), with primary action on the embryo. Higher doses given soon after implantation (day 5) induce early pregnancy termination by a primary effect on decidual tissue. Biochemical investigations in animals treated on day 5 or day 7 showed that DFMO induces a rapid and dramatic fall in ODC activity in the products of conception. These findings strengthen and extend previous evidence that ODC and polyamines play an essential role in the maintenance of early pregnancy in rodents.

Animals↗
PubMed1982

Evidence for a role of progesterone in the control of uterine ornithine decarboxylase in the pregnant hamster.

Changes in uterine ornithine decarboxylase (ODC) activity throughout pregnancy and the importance of progesterone in the regulation of this enzyme during the early post-implantation period have been studied in the hamster. Soon after implantation, from day 5 to day 6 of pregnancy, ODC activity rapidly increased. It reached a plateau on day 7, then abruptly fell on day 8 and remained low until the end of pregnancy. DL-alpha-difluoromethyl-ornithine (DFMO), an irreversible inhibitor of ODC, induced pregnancy termination as a consequence of the reduction of uterine ODC activity. When pregnancy arrest was induced by removing endogenous progesterone by administration of prostaglandin F2 alpha (PGF2 alpha) or by ovariectomy, the ODC rise was completely abolished, and exogenous progesterone was able to entirely counteract this effect on the enzyme activity and the termination of pregnancy. These results suggest that progesterone play a significant role in the rise of uterine ODC activity, which appears to be essential for the early post-implantation events needed for pregnancy maintenance.

Animals↗
PubMed1982

Lymphocyte subpopulations and surface membrane immunoglobulins in myasthenia gravis.

Lymphocyte subpopulations were studied in 16 myasthenic patients by means of E and EA7s rosettes and surface membrane immunofluorescence. Myasthenic patients showed an increase in lymphocytes with surface IgM an IgD when compared to a control group. In female patients this increase was highly significant, and lymphocytes bearing membrane IgG and IgA were also augmented. No difference was found in male patients when compared with sex-matched controls. These results suggest an activation of the B cell compartment restricted to female patients, and support the hypothesis of different immunopathogenetic mechanisms in this disease.

Adult↗
PubMed1981

Phosphoenolpyruvate carboxykinase (guanosine 5'-triphosphate) from rat liver cytosol. Divalent cation involvement in the decarboxylation reactions.

The presence of a divalent metal ion together with a catalytic amount of inosine 5'-diphosphate (IDP) is essential for the formation of pyruvate from oxalacetate catalyzed by purified rat liver cytosol phosphoenolpyruvate carboxykinase (PEPCK). With decreasing order of effectiveness, this pyruvate-forming activity was supported by micromolar levels of Cd2+, Zn2+, Mn2+, and Co2+. At the same concentrations, Mg2+ or Ca2+ was not effective. Combinations of Cd2+ with either Zn2+, Mn2+ or Co2+ were not additive with respect to the pyruvate-forming activity of PEPCK. Kinetic determination, with Cd2+ as the supporting cation, showed a 1:1 stoichiometry of interaction between each enzyme molecule and the nonconsumable substrate IDP. With 10 muM added Cd2+, the apparent Km for oxalacetate was 41 muM, and the apparent Ka for IDP was 0.25 muM. With Zn2+ or Mn2+, the apparent Ka for IDP was 0.2 or 0.13 muM, respectively. The effect of divalent transition-metal ions on PEPCK-catalyzed formation of phosphoenolpyruvate from oxalacetate was also investigated. Under steady-state conditions, the basal activity with MgITP was effectively enhanced with micromolar levels of Mn2+, Cd2+, or Co2+ included in the assay. The Vm increased 7- and 3.6-fold, and the apparent Km for MgITP changed by about a factor of 2 with the optimal concentrations of Mn2+ and Co2+, respectively. The most striking changes were in the apparent Km values for oxalacetate, which decreased to one-third and one-tenth when either Mn2+ or Co2+ was present in the assay together with Mg2+. The possible physiological importance of this kinetic effect is discussed.

Animals↗
PubMed1981

Thrombin inhibitors in women on oral contraceptives.

Progressive antithrombin activity and the immunological levels of antithrombin III, alpha 2-macroglobulin, and alpha 1-antitrypsin were measured in women at various intervals during treatment with oral contraceptives and in a group of untreated women. No significant changes were observed in any of the above parameters. In both control and treated women, there was a positive statistical correlation between progressive antithrombin activity and antithrombin III immunological levels, whereas no correlation was found between the former and alpha 1-antitrypsin or alpha 2-macroglobulin immunological levels. This study questions the possibility of thrombin inhibitors being selective signs of hypercoagulability during treatment with oral contraceptives.

Adolescent↗
PubMed1981

[An approach to hypertension in a native population].

In the article the authors illustrate: a) a community detection program designed to identify subjects with high blood pressure for treatment; b) the degree of participation; c) the cost of realization; d) the prevalence of arterial hypertension. A total of 4.149 subjects, or 65% of the examined population over age 8, were screened. The opportunity to have their blood pressure checked were offered with a letter of invitation to a centralized ambulatory. The cost of realization was L. 360 for every screened subject. The prevalence of hypertension was 20.4%.

Adolescent↗